Effect of nucleotide on the binding of N-N'-p-Phenylenedimaleimide-modified S-1 to unregulated and regulated actin

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Abstract
In our previous study [Chalovich, J. M., Greene, L. E., and Eisenberg, E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4909-4913], myosin subfragment 1 that was modified by having its 2 reactive thiol groups cross-linked by N,N''-p-phenylenedimaleimide (pPDM) that was found to resemble the myosin subfragment 1-adenosine 5''-triphosphate (S-1.cntdot.ATP) complex in its interaction with actin. In the present study, we examined the effect of actin on adenosine 5''-diphosphate (ADP) trapped at the active site of pPDM.cntdot.S-1. Our results indicate first that, in the presence of actin, ADP is no longer trapped at the active site but exchanges rapidly with free nucleotide. Different pPDM.cntdot.S-1-nucleotide complexes were then formed by exchanging nucleotide into the active site of pPDM.cntdot.S-1 in the presence of actin. The binding of pPDM.cntdot.S-1ATP or pPDM.cntdot.S-1.cntdot.PPi to actin is virtually identical with that of unmodified S-1 in the presence of ATP. Specifically, at .mu. = 18 mM, 25.degree. C, pPDM.cntdot.S-1.cntdot.ATP or pPDM.cntdot.S-1.cntdot.PPi binds to unregulated actin with the same affinity as does S-1.cntdot.ATP, and this binding does not appear to be affected by tropinin-tropomyosin. On the other hand, pPDM.cntdot.S-1.cntdot.ADP and pPDM.cntdot.S-1 with no bound nucleotide both show a small, but significant, difference between their binding to actin and the binding of S-1.cntdot.ATP; pPDM.cntdot.S-1 and pPDM.cntdot.S-1.cntdot.ADP both bind about 2- to 3-fold more strongly to unregulated actin than does S-1.cntdot.ATP. In addition, troponin-tropomyosin confers slight cooperative strengthening and Ca2+ sensitivity on the binding of pPDM.cntdot.S-1 and pPDM.cntdot.S-1.cntdot.ADP to actin. These results suggest that the nucleotide bound to the active site of pPDM.cntdot.S-1 causes conformational changes in the protein which, in turn, subtly alter is interaction with actin; the stronger the binding of the S-1 to unregulated actin, the more cooperativity is observed in its binding to regulated actin.

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